Treating hair by targeting enzymes

ABSTRACT

The invention provides the use of a hair benefit agent as an ingredient in a topical hair treatment composition for the purpose of targeting an active endogenous hair fiber enzyme when the composition is applied to the hair, thereby delivering a hair benefit via the interaction of the hair benefit agent with the enzyme.

This is a divisional of Ser. No. 09/553,634 filed Apr. 20, 2000, nowabandoned.

FIELD OF THE INVENTION

This invention relates to the treatment of hair with activators,inhibitors, co-factors or catalytic substrates for active endogenoushair fibre enzymes.

BACKGROUND AND PRIOR ART

The vast number of biochemical reactions that occur in every human cellare nearly all mediated by enzymes. Enzymes are biological catalysts,facilitating reactions under mild and physiological conditions. Allenzymes have a defined function and are highly specific for thereactions they catalyse. A particular enzyme could therefore be targetedby a specific reagent, whilst other enzymes remain unaffected. Inaddition, the types of reactions catalysed by different enzymes isextremely variable, and each enzyme is capable of catalysing up to amillion reaction events per second.

Enzymes are of particular interest for the development of new modes ofbenefit delivery in hair care. For example, U.S. Pat. No. 5,490,980describes a composition for topical application to skin, hair or nailscontaining a beneficial active agent which has been functionalised withan alkylamine moiety, together with the enzyme transglutaminase (acalcium and thiol dependent enzyme responsible for the crosslinking ofproteins by the formation of covalent bonds between lysine and glutamineresidues). The transglutaminase in the composition is said to act as acatalyst to crosslink the active ingredient with glutamine residues inskin, hair or nails. The transglutaminase in the composition of U.S.Pat. No. 5,490,980 is sourced from guinea pig liver, slime mould,alfalfa or preferably bacterial fermentation.

Problems with the above approach include size exclusion of the exogenousenzyme, the expense of obtaining the exogenous enzyme, the possibilityof immunological sensitisation from enzyme-containing formulations, andinstability of the enzyme when stored in the formulation, particularlywhere high surfactant levels are present as in cleansing compositions.Furthermore, Gardner et al. (1995) J. Soc. Cosmet. Chem., 46, 11-28provides evidence that glutamine residues on the surface of human hairare not, contrary to previous thought, recognised as a substrate forguinea pig liver transglutaminase. The authors of that paper found noconclusive evidence that virgin hair was modified by exogenously appliedtransglutaminase, and considered that the outer fatty acid layer of thehair probably restricted access to candidate glutamine sites, despitethe large abundance of glutamine residues in hair, through hydrophobicrepulsion or steric interactions. It was suggested that future workshould be directed to cross-linking soluble protein or modified proteinfilms co-deposited on hair together with the exogenous enzyme, so thatthe hair was not required to donate endogenous residues to the reaction.

Tsushima et al., Arch. Dermatol. Res. 284: 380-385 (1992) describes thepurification and characterisation of a cystatin-type cysteine proteinaseinhibitor (CPI) in the human hair shaft. In that paper, it is speculatedthat there are cysteine proteinases in hair, since there is CPI.However, the presence of any such enzymes within the hair fibre has, todate, not been reported in the literature.

The inventors have now found that the mature human hair fibre containsendogenous enzymes, which furthermore have been shown to be active andtherefore capable of interaction with exogenously supplied substrates.The precise origin of these active endogenous hair fibre enzymes remainsunclear. It is particularly surprising that these endogenous hair fibreenzymes have not only been shown by the inventors to be present, butalso to be biologically active. Maturation of hair fibre results in thedeath of its constituent cells (Tamada et al. (1994) Br. J. Dermatol.131: 521-524) and this coupled with the increased levels ofintracellular cross-linking results in a mature fibre which ismetabolically dead. Unexpectedly, the inventors have found that enzymeactivity is in fact preserved, rather than denatured, during theprocesses of cellular keratinisation and death that occur during fibregrowth.

SUMMARY OF THE INVENTION

The present invention provides the use of a hair benefit agent as aningredient in a topical hair treatment composition for the purpose oftargeting an active endogenous hair fibre enzyme when the composition isapplied to the hair, thereby delivering a hair benefit via theinteraction of the hair benefit agent with the enzyme.

Advantageously, the above approach solves the problems associated withthe systems described previously involving application of exogenousenzyme. For example, enzyme substrates can be selected which arecheaper, safer and stabler to formulate into hair treatment compositionsthan exogenous enzyme, and which, unlike exogenous enzyme, are able topenetrate the hair and actually deliver enzyme-linked benefits to theunderlying fibre matrix.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

Examples of active endogenous enzymes identified by the inventors todate within the mature human hair fibre fall in various and diverseclasses and include:

Transglutaminase

Protease

Lipase

Steroid sulphatase

Catalase

Esterase

Hair Benefit Agents

Ingredients suitable for use as hair benefit agents according to theinvention for targeting endogenous hair fibre enzymes may be anymaterial which is capable of specifically interacting with the selectedenzyme, or is chemically modified in order to do so. The material mayserve as an enzyme activator, enzyme inhibitor, enzyme co-factor orcatalytic substrate for the enzyme, in order to derive benefit.

Examples of the type of such ingredients include hair conditioningagents, (such as humectants, softeners and cuticle lubricants), haircolouring agents, antimicrobial compounds, UV-absorbing compounds,fluorescers, hair strengthening agents (such as fibre repair agents orfibre rebuilding agents), antioxidants, perfumes, and mixtures thereof.

Preferred ingredients for use according to the invention are selected totarget the endogenous hair fibre enzymes which are classified below:

(i) Transglutaminase

Transglutaminase has been found to be active in the hair fibre,localised predominantly in the cuticle. It is a calcium and thioldependent enzyme which is responsible for the crosslinking of proteinsby the formation of covalent bonds between lysine and glutamineresidues.

Ingredients suitable for use as hair benefit agents according to theinvention for targeting endogenous hair fibre transglutaminase includecatalytic substrates for this class of enzyme.

Catalytic substrates for transglutaminase are required to include (or bemodified to include) at least one —R′NH₂ group in which R′ is ahydrocarbon or functionalised hydrocarbon chain. Preferably R′ is astraight aliphatic hydrocarbon chain containing from 1 to 8 carbonatoms.

Transglutaminase crosslinks the substrate through the alkyl amine(R′NH₂) group to the glutamine residues in hair.

Accordingly, hair benefit agents suitable for use according to theinvention as catalytic substrates for endogenous hair transglutaminasemay be any compound which has a beneficial effect when delivered tohuman hair, as long as the compound contains, or is modified to contain,an alkyl amine group.

Use of such hair benefit agents as catalytic substrates for endogenoushair fibre transglutaminase enables a direct, in situ interactionbetween substrate and enzyme for delivering a localised and sustainedhair fibre benefit derived from that interaction. In this way, permanentor internal hair benefits may be obtained from thetransglutaminase-catalysed crosslinking reaction between a benefit agentand the substance of the hair fibre itself. Preferred benefit agents inthis context are hair conditioning agents and hair colouring agents.Advantageously, such enzyme-catalysed interactions occur underphysiological and naturally mild reaction conditions, so harsh chemicaltreatments are not necessary.

Preferably, in order to optimise the crosslinking reaction betweentransglutaminase and substrate, the R′NH₂ group contains at least 4unbranched carbon atoms adjacent to the NH₂ group. Optimaltransglutaminase activity is generally achieved when the substrate has(a) alkylamine side-chain lengths equivalent to 5 methylene groups (or7.2-7.6 A long), (b) no branching nor groups bulkier than methylenealong the alkyl amine chain, (c) hydrophobic moieties attached to thealkyl chain, and (d) more than one alkyl amine group. However, this isnot particularly critical and other hydrocarbon chains can be used,(including those incorporating functional groups such as ester, ether oramide linkages or similar), provided the terminal amine group is notsterically hindered from interacting with the active site.

Suitable ingredients which inherently contain an alkyl amine groupinclude intact proteins, protein hydrolysates, chemically modified (e.g.quaternized or acylated) proteins or protein hydrolysates, peptides,non-proteinaceous amino acid polymers (such as amino acid polymersproduced by chemical synthesis), amino acids (or derivatives thereof),and primary amine compounds.

Preferred examples include the amino acid lysine, polymers thereof(polylysine) having a molecular weight ranging from 100 to 2,000, andacylated derivatives thereof such as lauroyllysine.

Also preferred are primary amines having a carbon chain of at least C₁₀,preferably C₁₂₋₂₂, such as soya amine, hydrogenated tallow amine,stearyl amine, tallow amine, oleyl amine, hexadecylamine,octadecylamine, and combinations thereof. These materials are preferredsince they are capable of imparting smoothness and improved ease ofcombing to treated hair fibres.

Ingredients may also be used which have a beneficial effect on humanhair and which have been modified to contain at least one alkyl aminemoiety. U.S. Pat. No. 5,490,980 describes and exemplifies a range ofsuch ingredients and the corresponding process by which they areappropriately modified. Preferred examples are alkyl amine modifiedsilicones which are capable of imparting conditioning benefits to hair.

Activators of endogenous hair fibre transglutaminase may also be used asingredients according to the invention. Such activators may be usedalone, in order to promote fibre strengthening due to enhanced in situfibre transglutaminase activity, or alternatively in conjunction withone or more of the catalytic substrates described above.

(ii) Protease

Various protease activities have been identified in the hair fibre.These include cysteine and serine proteases, with the major proteaseobserved to date appearing to have similar properties to the lysosomalcysteine proteases, cathepsins L and B. Cathepsins L and B are known tohydrolyse many cytosolic and structural proteins, including collagen andelastin.

Ingredients suitable for use as hair benefit agents according to theinvention for targeting endogenous hair fibre protease includeinhibitors of this class of enzyme. Examples of such protease inhibitorsinclude cystatins, which are small proteins found widely in nature, forexample in eggs and avocado mesocarp. Use of such inhibitors enablesreduction or prevention of autohydrolysis of the hair fibre by theactive hair fibre protease, thereby protecting against hair fibre damageor deterioration.

Further suitable ingredients for use as hair benefit agents according tothe invention for targeting endogenous hair fibre protease are catalyticsubstrates for protease, i.e. protein, proteinaceous or peptidecontaining substrates capable of being hydrolysed by the protease intosmaller peptides and/or free amino acids.

Examples include collagen, keratin, fibroin, elastin, ovalbumin, casein,gluten, ferritin, gliadin, zein, soy protein, silk protein andhydrolysates or derivatives thereof.

Preferably the substrate is rich in the amino acids lysine, histidineand/or arginine. In this way, hydrolysis of substrate by the enzyme candeliver benefits such as moisturisation to the hair via the release ofthe free amino acids directly into the hair fibre. For example,preferred protein or proteinaceous substrates contain at least 12%, mostpreferably at least 15% to about 70% of these amino acids, either singlyor in admixture, by weight based on total weight.

Ingredients may also be used which have a beneficial effect on humanhair and which have been covalently modified to contain an amino acid oramino acid sequence capable of interacting with endogenous air fibreprotease. In this way, protease-mediated catalysis facilitates thegradual release of benefit agent directly into the hair fibre, where itmay be allowed to penetrate further into the fibre, without the need forharsh chemical modification processes.

(iii) Lipase

Lipase activity has been found in the hair fibre. Lipases hydrolyseester linkages in lipids, and in particular those withintriacylglycerol. This reaction yields free fatty acids and glycerol.Glycerolipids are the most common substrates that are hydrolysed by theenzyme, although they are relatively non-specific and will alsohydrolyse other acyl esters. Lipases are believed to have an importantrole in hair development where they are involved in the recycling ofhair glycerolipids during the formation of cell membrane complex lipids.During hair cell differentiation, the cellular lipids (predominantlyglycerolipids) are destroyed and new lipids synthesised such assteroids, ceramides and fatty acids.

Ingredients suitable for use as hair benefit agents according to theinvention for targeting endogenous hair fibre lipase include catalyticsubstrates for this class of enzyme.

Examples are triglyceride fats and oils derived from vegetable, animaland marine sources such as coconut oil, castor oil, safflower oil,cotton seed oil, corn oil, olive oil, cod liver oil, almond oil, avocadooil, palm oil, sesame oil, and soybean oil.

Preferred are triglycerides derived from fatty acids having 8 to 22carbon atoms. Suitable examples of such fatty acids include pelargonic,lauric, myristic, palmitic, stearic, isostearic, 12-hydroxystearic,oleic, linoleic, ricinoleic, 18-methyleicosanoic, arachidonic, behenic,and erucic acids. In this way, lipase-mediated catalysis facilitates therelease of free fatty acids, which are important components of the cellmembrane complex, and glycerol, an effective humectant, directly intothe hair fibre. Advantageously, targeting of endogenous hair fibrelipase with triglyceride substrates of selected fatty acid contentprovides a route to influencing the lipid environment of the hair fibrewithout the necessity for harsh chemical or physical modificationprocesses.

Ingredients may also be used which have a beneficial effect on humanhair and which have been covalently tagged with a lipid capable ofinteracting with endogenous hair fibre lipase.

(iv) Steroid Sulphatase

Steroid sulphatase activity has been identified in the hair fibre. Thisenzyme catalyses the hydrolysis of cholesterol-3-sulphate intocholesterol (and sulphate). Steroid sulphatase is believed to have animportant role in the formation of normal healthy hair, since dramatichair effects are noticeable in people who are deficient in this enzyme.For example, in patients with X-linked ichthiosis, the enzyme activityis reduced (or non-existent) and the hair feels dry and brittle.

Ingredients suitable for use as hair benefit agents according to theinvention for targeting endogenous hair fibre steroid sulphatase includeactivators of this class of enzyme. Use of such activators enablesincreased delivery of cholesterol directly into the hair fibre.Cholesterol has been shown to protect hair against damage, enhance hairflexibility and reduce dry feel.

Further suitable ingredients for use as hair benefit agents according tothe invention for targeting endogenous hair fibre steroid sulphatase arecatalytic substrates for steroid sulphatase such ascholesterol-3-sulphate, which is capable of being hydrolysed by thesteroid sulphatase into cholesterol (and sulphate).

(v) Catalase

Catalase activity has been identified in the hair fibre. This enzymecatalyses the conversion of hydrogen peroxide (H₂O₂) into water andmolecular oxygen.

Ingredients suitable for use as hair benefit agents according to theinvention for targeting endogenous hair fibre catalase includeactivators of this class of enzyme.

Use of such activators provides a route to additional radical damageprotection for the hair fibre through increased removal of peroxide.This is particularly advantageous in the case of chemically treated hair(e.g. bleached, coloured or permed) which is especially vulnerable tooxidative damage, and also in climates in which high levels of oxidativestress are encountered.

Use of such activators additionally provides a route to removal ofsurplus peroxide which is deliberately added to the hair duringtreatment with products such as bleaches, colorants, and perms in whichperoxide is used for the oxidation step. Alternatively, inhibitors ofendogenous hair fibre catalase may be used as hair benefit agentsaccording to the invention, as ingredients for increasing the efficacyof peroxide-containing products which are applied to the hair fibre suchas colourants, bleaches or perms.

(vi) Esterase

Esterase activity has been identified in the hair fibre. Esterases are abroad class of enzymes that catalyse the hydrolysis of ester linkages.

Ingredients suitable for use as hair benefit agents according to theinvention for targeting endogenous hair fibre esterase include catalyticsubstrates for this class of enzyme.

Catalytic substrates for esterase are required to include (or bemodified to include) at least one ester linkage.

Accordingly, hair benefit agents suitable for use according to theinvention as catalytic substrates for endogenous hair esterase may beany compound which has a beneficial effect when delivered to human hair,as long as the compound contains, or is modified to contain, an esterlinkage.

In this way, benefit agents may be used in esterified form, (forexample, conjugated via an ester linkage to an inert carrier molecule),and used to target endogenous hair fibre esterase, so that uponhydrolysis by endogenous hair fibre esterase, the benefit agent isreleased and thus activated, allowing a controlled release of thebenefit agent directly into the hair fibre. This is particularlyadvantageous in the case of benefit agents which in free (unesterified)form are sensitive to factors such as heat, oxidation, light, moisture,pH, or microbial attack, or which tend to react with other ingredientsin a formulation. Examples of such benefit agents include perfumes,colourants, antioxidants such as vitamins A, C and E, and natural hairnutrients such as lipids and amino acids.

Product Form

The final product form of hair treatment compositions according to theinvention may suitably be, for example, shampoos, conditioners, sprays,mousses or lotions. Particularly preferred product forms are shampoos,post-wash conditioners (leave-in and rinse-off) and hair treatmentproducts such as hair essences and hair oils.

Shampoo Compositions

A particularly preferred hair treatment composition in accordance withthe invention is a shampoo composition.

Cleansing Surfactant

Such a shampoo composition will comprise one or more cleansingsurfactants which are cosmetically acceptable and suitable for topicalapplication to the hair. Further surfactants may be present as anadditional ingredient if sufficient for cleansing purposes is notprovided as emulsifying agent for oily or hydrophobic components (suchas silicones) which may typically be present in the shampoo.

It is preferred that shampoo compositions of the invention comprise atleast one further surfactant (in addition to that used as emulsifyingagent) to provide a cleansing benefit.

Suitable cleansing surfactants, which may be used singularly or incombination, are selected from anionic, amphoteric and zwitterionicsurfactants, and mixtures thereof. The cleansing surfactant may be thesame surfactant as the emulsifier, or may be different.

Examples of anionic surfactants are the alkyl sulphates, alkyl ethersulphates, alkaryl sulphonates, alkanoyl isethionates, alkyl succinates,alkyl sulphosuccinates, N-alkyl sarcosinates, alkyl phosphates, alkylether phosphates, alkyl ether carboxylates, and alpha-olefinsulphonates, especially their sodium, magnesium, ammonium and mono-, di-and triethanolamine salts. The alkyl and acyl groups generally containfrom 8 to 18 carbon atoms and may be unsaturated. The alkyl ethersulphates, alkyl ether phosphates and alkyl ether carboxylates maycontain from 1 to 10 ethylene oxide or propylene oxide units permolecule.

Typical anionic surfactants for use in shampoos of the invention includesodium oleyl succinate, ammonium lauryl sulphosuccinate, ammonium laurylsulphate, sodium dodecylbenzene sulphonate, triethanolaminedodecylbenzene sulphonate, sodium cocoyl isethionate, sodium laurylisethionate and sodium N-lauryl sarcosinate. The most preferred anionicsurfactants are sodium lauryl sulphate, triethanolamine monolaurylphosphate, sodium lauryl ether sulphate 1EO, 2EO and 3EO, ammoniumlauryl sulphate and ammonium lauryl ether sulphate 1EO, 2EO and 3EO.

Examples of amphoteric and zwitterionic surfactants include alkyl amineoxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulphobetaines(sultaines), alkyl glycinates, alkyl carboxyglycinates, alkylamphopropionates, alkylamphoglycinates, alkyl amidopropylhydroxysultaines, acyl taurates and acyl glutamates, wherein the alkyland acyl groups have from 8 to 19 carbon atoms. Typical amphoteric andzwitterionic surfactants for use in shampoos of the invention includelauryl amine oxide, cocodimethyl sulphopropyl betaine and preferablylauryl betaine, cocamidopropyl betaine and sodium cocamphopropionate.

The shampoo composition can also include co-surfactants, to help impartaesthetic, physical or cleansing properties to the composition. Apreferred example is a nonionic surfactant, which can be included in anamount ranging from 0% to about 5% by weight based on total weight. Forexample, representative nonionic surfactants that can be included inshampoo compositions of the invention include condensation products ofaliphatic (C₈-C₁₈) primary or secondary linear or branched chainalcohols or phenols with alkylene oxides, usually ethylene oxide andgenerally having from 6 to 30 ethylene oxide groups.

Other representative nonionics include mono- or di-alkyl alkanolamides.Examples include coco mono- or di-ethanolamide and cocomono-isopropanolamide.

Further nonionic surfactants which can be included in shampoocompositions of the invention are the alkyl polyglycosides (APGs).Typically, the APG is one which comprises an alkyl group connected(optionally via a bridging group) to a block of one or more glycosylgroups. Preferred APGs are defined by the following formula:

RO—(G)_(n)

wherein R is a branched or straight chain alkyl group which may besaturated or unsaturated and G is a saccharide group.

R may represent a mean alkyl chain length of from about C₅ to about C₂₀.Preferably R represents a mean alkyl chain length of from about C₈ toabout C₁₂. Most preferably the value of R lies between about 9.5 andabout 10.5. G may be selected from C₅ or C₆ monosaccharide residues, andis preferably a glucoside. G may be selected from the group comprisingglucose, xylose, lactose, fructose, mannose and derivatives thereof.Preferably G is glucose.

The degree of polymerisation, n, may have a value of from about 1 toabout 10 or more. Preferably, the value of n lies in the range of fromabout 1.1 to about 2. Most preferably the value of n lies in the rangeof from about 1.3 to about 1.5.

Suitable alkyl polyglycosides for use in the invention are commerciallyavailable and include for example those materials identified as: OramixNS10 ex Seppic; Plantaren 1200 and Plantaren 2000 ex Henkel.

The total amount of surfactant (including any co-surfactant, and/or anyemulsifying agent) in shampoo compositions of the invention is generallyfrom 0.1 to 50% by weight, preferably from 5 to 30%, more preferablyfrom 10% to 25% by weight of the total shampoo composition.

Cationic Polymer

A cationic polymer is a preferred ingredient in shampoo compositions ofthe invention, for enhancing conditioning performance of the shampoo.Typically such a polymer enhances deposition of conditioning componentssuch as silicone from the shampoo composition onto the intended siteduring use, i.e. the hair and/or the scalp.

The cationic polymer may be a homopolymer or be formed from two or moretypes of monomers. The molecular weight of the polymer will generally bebetween 5000 and 10000000, typically at least 10000 and preferably inthe range 100000 to about 2000000. The polymers will have cationicnitrogen containing groups such as quaternary ammonium or protonatedamino groups, or a mixture thereof.

The cationic nitrogen-containing group will generally be present as asubstituent on a fraction of the total monomer units of the cationicpolymer. Thus when the polymer is not a homopolymer it can containspacer non-cationic monomer units. Such polymers are described in theCTFA Cosmetic Ingredient Directory, 3rd edition. The ratio of thecationic to non-cationic monomer units is selected to give a polymerhaving a cationic charge density in the required range.

Suitable cationic polymers include, for example, copolymers of vinylmonomers having cationic amine or quaternary ammonium functionalitieswith water soluble spacer monomers such as (meth)acrylamide, alkyl anddialkyl (meth)acrylamides, alkyl (meth)acrylate, vinyl caprolactone andvinyl pyrrolidine. The alkyl and dialkyl substituted monomers preferablyhave C1-C7 alkyl groups, more preferably C1-3 alkyl groups. Othersuitable spacers include vinyl esters, vinyl alcohol, maleic anhydride,propylene glycol and ethylene glycol.

The cationic amines can be primary, secondary or tertiary amines,depending upon the particular species and the pH of the composition. Ingeneral secondary and tertiary amines, especially tertiary, arepreferred.

Amine substituted vinyl monomers and amines can be polymerised in theamine form and then converted to ammonium by quaternization.

The cationic polymers can comprise mixtures of monomer units derivedfrom amine- and/or quaternary ammonium-substituted monomer and/orcompatible spacer monomers.

Suitable cationic polymers include, for example:

copolymers of 1-vinyl-2-pyrrolidine and 1-vinyl-3-methyl-imidazoliumsalt (e.g. chloride salt), referred to in the industry by the Cosmetic,Toiletry, and Fragrance Association, (CTFA) as Polyquaternium-16. Thismaterial is commercially available from BASF Wyandotte Corp.(Parsippany, N.J., USA) under the LUVIQUAT tradename (e.g. LUVIQUAT FC370);

copolymers of 1-vinyl-2-pyrrolidine and dimethylaminoethyl methacrylate,referred to in the industry (CTFA) as Polyquaternium-11. This materialis available commercially from Gaf Corporation (Wayne, N.J., USA) underthe GAFQUAT tradename (e.g., GAFQUAT 755N);

cationic diallyl quaternary ammonium-containing polymers including, forexample, dimethyldiallyammonium chloride homopolymer and copolymers ofacrylamide and dimethyldiallylammonium chloride, referred to in theindustry (CTFA) as Polyquaternium 6 and Polyquaternium 7, respectively;

mineral acid salts of amino-alkyl esters of homo-and copolymers ofunsaturated carboxylic acids having from 3 to 5 carbon atoms, (asdescribed in U.S. Pat. No. 4,009,256);

cationic polyacrylamides(as described in WO95/22311).

Other cationic polymers that can be used include cationic polysaccharidepolymers, such as cationic cellulose derivatives, cationic starchderivatives, and cationic guar gum derivatives.

Cationic polysaccharide polymers suitable for use in compositions of theinvention include those of the formula:

A—O—[R—N⁺(R¹)(R²)(R³)X⁻],

wherein: A is an anhydroglucose residual group, such as a starch orcellulose anhydroglucose residual. R is an alkylene, oxyalkylene,polyoxyalkylene, or hydroxyalkylene group, or combination thereof. R¹,R² and R³ independently represent alkyl, aryl, alkylaryl, arylalkyl,alkoxyalkyl, or alkoxyaryl groups, each group containing up to about 18carbon atoms. The total number of carbon atoms for each cationic moiety(i.e., the sum of carbon atoms in R¹, R² and R³) is preferably about 20or less, and X is an anionic counterion.

Cationic cellulose is available from Amerchol Corp. (Edison, N.J., USA)in their Polymer JR (trade mark) and LR (trade mark) series of polymers,as salts of hydroxyethyl cellulose reacted with trimethyl ammoniumsubstituted epoxide, referred to in the industry (CTFA) asPolyquaternium 10. Another type of cationic cellulose includes thepolymeric quaternary ammonium salts of hydroxyethyl cellulose reactedwith lauryl dimethyl ammonium-substituted epoxide, referred to in theindustry (CTFA) as Polyquaternium 24. These materials are available fromAmerchol Corp. (Edison, N.J., USA) under the tradename Polymer LM-200.

Other suitable cationic polysaccharide polymers include quaternarynitrogen-containing cellulose ethers (e.g. as described in U.S. Pat. No.3,962,418), and copolymers of etherified cellulose and starch (e.g. asdescribed in U.S. Pat. No. 3,958,581).

A particularly suitable type of cationic polysaccharide polymer that canbe used is a cationic guar gum derivative, such as guarhydroxypropyltrimonium chloride (Commercially available fromRhone-Poulenc in their JAGUAR trademark series).

Examples are JAGUAR C13S, which has a low degree of substitution of thecationic groups and high viscosity. JAGUAR C15, having a moderate degreeof substitution and a low viscosity, JAGUAR C17 (high degree ofsubstitution, high viscosity), JAGUAR C16, which is a hydroxypropylatedcationic guar derivative containing a low level of substituent groups aswell as cationic quaternary ammonium groups, and JAGUAR 162 which is ahigh transparency, medium viscosity guar having a low degree ofsubstitution.

Preferably the cationic polymer is selected from cationic cellulose andcationic guar derivatives. Particularly preferred cationic polymers areJAGUAR C13S, JAGUAR C15, JAGUAR C17 and JAGUAR C16 and JAGUAR C162.

Conditioners

Conditioning Surfactant

Compositions in accordance with the invention may also be formulated asconditioners for the treatment of hair (typically after shampooing) andsubsequent rinsing.

Such a conditioner will comprise one or more conditioning surfactantswhich are cosmetically acceptable and suitable for topical applicationto the hair.

Suitable conditioning surfactants are selected from cationicsurfactants, used singly or in admixture.

Examples of suitable cationic conditioning surfactants includequaternary ammonium cationic surfactants.

Suitable quaternary ammonium cationic surfactants for use in hairconditioners of the invention include cetyltrimethylammonium chloride,behenyltrimethylammonium chloride, cetylpyridinium chloride,tetramethylammonium chloride, tetraethylammonium chloride,octyltrimethylammonium chloride, dodecyltrimethylammonium chloride,hexadecyltrimethylammonium chloride, octyldimethylbenzylammoniumchloride, decyldimethylbenzylammonium chloride,stearyldimethylbenzylammonium chloride, didodecyldimethylammoniumchloride, dioctadecyldimethylammonium chloride, tallowtrimethylammoniumchloride, cocotrimethylammonium chloride, and the correspondinghydroxides thereof. Further suitable cationic surfactants include thosematerials having the CTFA designations Quaternium-5, Quaternium-31 andQuaternium-18. Mixtures of any of the foregoing materials may also besuitable. A particularly useful cationic surfactant for use in hairconditioners of the invention is cetyltrimethylammonium chloride,available commercially, for example as GENAMIN CTAC, ex HoechstCelanese.

A further preferred class of cationic conditioning surfactants areacid-neutralised amidoamine compounds of the general structural formula(I):

R1—C(O)—NH—R2—N(R3)(R4)  (I)

wherein R1 is a fatty acid chain containing from 12 to 22 carbon atoms,R2 is an alkylene group containing from one to four carbon atoms, and R3and R4 are, independently, an alkyl group having from one to four carbonatoms.

Examples of suitable amidoamine compounds of general structural formula(I) include stearamidopropyl dimethylamine, stearamidopropyldiethylamine, stearamidoethyl dimethylamine, stearamidoethyldiethylamine, palmitamidopropyl dimethylamine, behenamidopropyldimethylamine, myristamidopropyl dimethylamine, oleamidopropyldimethylamine, ricinoleamidopropyl dimetnylamine, and combinationsthereof.

The acid used to neutralise the amidoamine compound can be essentiallyany organic acid or mineral acid of sufficient acid strength toneutralise a free amine nitrogen. Such acids include hydrochloric acid,sulphuric acid, nitric acid, phosphoric acid, lactic acid, citric acid,tartaric acid, acetic acid, gluconic acid, glycolic acid and propionicacid, or combinations thereof. A preferred acid is lactic acid, sinceneutralisation of the amidoamine compound with this acid yields anexceptionally stable composition.

In general, a sufficient amount of acid is added to neutralise theamidoamine compound and to adjust the final pH of the composition towithin a range of from about 2.5 to about 6, preferably in a pH range offrom about 3 to about 5.

In conditioners of the invention, the level of cationic surfactant ispreferably from 0.01 to 10%, more preferably 0.05 to 5%, most preferably0.1 to 2% by total weight of cationic surfactant based on the totalweight of the composition.

Fatty Alcohol

Conditioners of the invention advantageously incorporate a fattyalcohol. The combined use of fatty alcohols and cationic surfactants inconditioning compositions is believed to be especially advantageous,because this leads to the formation of a lamellar phase, in which thecationic surfactant is dispersed.

Representative fatty alcohols comprise from 8 to 22 carbon atoms, morepreferably 16 to 20. Examples of suitable fatty alcohols include cetylalcohol, stearyl alcohol and mixtures thereof. The use of thesematerials is also advantageous in that they contribute to the overallconditioning properties of compositions of the invention.

The level of fatty alcohol in conditioners of the invention isconveniently from 0.01 to 10%, preferably from 0.1 to 5% by weight ofthe composition. The weight ratio of cationic surfactant to fattyalcohol is suitably from 10:1 to 1:10, preferably from 4:1 to 1:8,optimally from 1:1 to 1:4.

Silicone

Silicone is a particularly preferred ingredient in hair treatmentcompositions of the invention. In particular, hair shampoos andconditioners of the invention will preferably also comprise emulsifiedparticles of silicone, for enhancing conditioning performance. Thesilicone is insoluble in the aqueous matrix of the composition and so ispresent in an emulsified form, with the silicone present as dispersedparticles.

Suitable silicones include polydiorganosiloxanes, in particularpolydimethylsiloxanes which have the CTFA designation dimethicone. Alsosuitable for use compositions of the invention (particularly shampoosand conditioners) are polydimethyl siloxanes having hydroxyl end groups,which have the CTFA designation dimethiconol, and polydimethylsiloxaneshaving containing at least one amino functional group, which have theCTFA designation amodimethicone or trimethylsilylamodimethicone.

Examples of suitable materials include:

the DC200 series of silicone fluids, available from Dow Corning (e.g.DC200, viscosity 350 cs), or SF96 or the VISCASIL series of silicones,available from General Electric Silicones;

silicone gums such as SE30, SE54 and SE76, available from GeneralElectric Silicones;

silicone gum/fluid blends such as Q2-1403 available from Dow Corning, orCF 1251, available from General Electric Silicones;

pre-formed emulsions of dimethiconol such as emulsions DC2-1766,DC2-1784, DC2-1787 and microemulsions DC2-1391, DC2-1865 and DC2-1870,all available from Dow Corning;

amino functional silicones such as Q2-8220 and Q2-8466 fluids, availablefrom Dow Corning, and also SF-1708-D1, available from General ElectricSilicones;

pre-formed emulsions of amino functional silicones such as DC929Cationic Emulsion, DC939 Cationic Emulsion, and the non-ionic emulsionsDC2-7224, DC2-8467, DC2-8177 and DC2-8154 (all available from DowCorning);

silicone gum/silicone fluid/amino functional silicone blends.

Most preferably, the silicone for inclusion in the hair treatmentcomposition of the invention, is sourced as a pre-formed aqueousemulsion, for example a mechanically-formed aqueous emulsion. In suchemulsions, it is highly preferable that the emulsion additionallyincludes at least one emulsifier in order to stabilise the siliconeemulsion. Suitable emulsifiers are well known in the art and includeanionic and nonionic surfactants.

Preferably, the average particle size of the silicone droplets in theemulsion and also in the final hair treatment composition is less than20 microns, more preferably less than 10 microns. A smaller siliconeparticle size enables a more uniform distribution of silicone on thehair for the same amount of silicone in the composition.

Silicone particle size in the emulsion may be measured by means of alaser light scattering technique, for example using a 2600D ParticleSizer from Malvern Instruments.

Viscosity (of the silicone itself and not the emulsion or the final hairtreatment composition) can be measured by means of a glass capillaryviscometer as set out further in Dow Corning Corporate Test MethodCTM004, Jul. 20, 1970.

The total amount of silicone incorporated into hair treatmentcompositions of the invention depends on the level of conditioningdesired and the material used. A preferred amount is from 0.01 to about10% by weight of the total composition although these limits are notabsolute. The lower limit is determined by the minimum level to achieveconditioning and the upper limit by the maximum level to avoid makingthe hair and/or skin unacceptably greasy.

We have found that a total amount of silicone of from 0.3 to 5%,preferably 0.5 to 3%, by weight of the total composition is a suitablelevel.

Solvents and Carriers

Compositions of the invention are preferably aqueous based, butnon-aqueous solvents also can be used in order to help solubiliseingredients that are not sufficiently soluble in water. Suitablenon-aqueous solvents include the lower alcohols like ethyl alcohol andpropyl alcohol; polyols like glycerol; glycols or glycol ethers, like2-butoxyethanol, ethylene glycol, ethylene glycol monoethyl ether,propylene glycol and diethylene glycol monoethyl ether or monomethylether and mixtures thereof.

Optional Ingredients

Compositions of this invention may contain any other ingredient normallyused in hair treatment formulations. These other ingredients may includeviscosity modifiers, preservatives, colouring agents, polyols such asglycerine and polypropylene glycol, chelating agents, antioxidants,fragrances, antimicrobials and sunscreens. Each of these ingredientswill be present in an amount effective to accomplish its purpose.Generally these optional ingredients are included individually at alevel of up to about 5% by weight of the total composition.

The invention is further illustrated by way of the followingnon-limitative examples. All percentages quoted are by weight based ontotal weight unless otherwise stated.

EXAMPLES Example 1

Determination of the Presence of Steroid Sulphatase in Hair Fibres

Hair fibres taken from approx. 5 mm from the scalps of male individuals,aged 25-35, were cleaned, dried and then ground in liquid N₂, andhomogenised with an Ystral Homogeniser (high shear) using a 5 mm probein imidazole-HCl (pH 7), 1M NaCl and 0.1% Triton X-100. Sucrose extractswere prepared in a similar way to the above in 250 mM sucrose.

Both homogenates were centrifuged at 13000 rpm for 5 minutes. Bothsupernatant and insoluble proteinaceous pellet were retained. Thepellets were resuspended in 20% glycerol in 100 mM imidazole-HCl (pH 7).The clear supernatants (imidazole and sucrose extracts) were dialysedagainst 20% glycerol in 100 mM imidazole-HCl (pH 7). The extracts werefrozen at −20° C. until required.

Cholesterol-sulphate sulphatase activity of the hair fibre extracts wasmeasured using the following assay:

The assay mixture (per assay) contained 20 μM [¹⁴C]cholesterol-3-sulphate (250 000 dpm total) in 50 mM imidazole-HCl pH7,0.05% Triton X-100 and hair fibre extract in a final assay volume of 100μl. A typical assay was conducted by preparing a 5×substrate solution asfollows: 22.72 μl of [¹⁴C] cholesterol-3-sulphate was placed in a cleanglass vial and the solvent removed under a steady stream of N₂ gas. Thelipid was resuspended into 1 ml of 0.25% Triton X-100 in 250 mMimidazole-HCl (pH 7) and mixture vigorously mixed. The K_(m) of humansteroid sulphatase is 5 μM, and thus using 20 μM substrate in the assay,the velocity of the reaction would be tending towards maximum.

For each assay, 20 μl of the 5×substrate solution was added to 80 μl ofhair fibre extract, incubated at 37° C. for 3 hours, followed by up to21 hours at room temperature. The reaction was stopped by the additionof chloroform:methanol (2:1). This was then transferred to a clean vial,and any residual reaction mixture was reclaimed with two 500 μl washeswith the solvent. Milli-Q water (0.5 ml) was added to the pooled solventand vigorously mixed for 5 seconds and allowed to stand to enable thephases to separate. The lower (chloroform) phase was placed in a freshvial and the solvent removed under a steady stream of N2 gas. The driedlipid was resuspended in 50 μl of chloroform:methanol (2:1) and thewhole sample loaded onto a preparative silica TLC plate.

A line in the silica plate was scored at 13 cm from the origin, ensuringthat the solvent front migration was fixed. The TLC plates weredeveloped in chloroform:methanol:acetic acid (100:2:1), alongsideappropriate standards. Using this solvent system cholesterol-3-sulphateand cholesterol had Rf values of 0.27 and 0.74, respectively. From eachsample lane, the area corresponding to cholesterol was scraped from theTLC plate and the silica finings placed in a glass vial. To each vial250 μl Chloroform:methanol (2:1) was added and the vial gently agitated.Scintillation fluid (10 ml) was added, the samples mixed and theradioactivity measured by scintillation counting.

The results showed that steroid sulphatase activity was present in thehair fibre extracts, located primarily in the insoluble proteinfraction. A radioactive region corresponding to cholesterol was notobserved in control samples of pre-boiled hair fibre extract orsubstrate only respectively. Cholesterol production was linear for 24hours, and a concomitant linear decrease in cholesterol-3-sulphate wasobserved.

Using the same assay protocol as described above, steroid sulphataseactivity was also observed in samples of finely ground hair which hadnot been subjected to an extraction procedure.

Example 2

Determination of the Presence of Lipase in Hair Fibres

A hair fibre extract was prepared from finely ground hair in imidazolebuffer using the extraction procedure described above in Example 1. Theextract was stored in aliquots in the presence of 20% glycerol at −20°C. until required. All solutions were syringe filtered through a nylon0.2 μm filter in order to reduce micro-organism contamination.

Lipase activity of the hair fibre extract was measured using thefollowing assay

Tri [¹⁴C]oleoylglycerol (triacylglycerol, 56 mCi/mmol, 100 μCi/ml) wasfrom obtained from Amersham-Pharmacia Biotech. Lipase activity wasmeasured by following the formation of [1-¹⁴C]oleic acid, mono[1-¹⁴C]oleoylglycerol and di [1-¹⁴C]oleoylglycerol by TLC. A stocksolution (5×) of tri[1-¹⁴C]oleoylglycerol in 250 mM Imidazole-HCl (pH 7)containing 0.25% Triton X-100 was prepared and 20 μl of this added to 80μl of hair enzyme extract. The reactions were incubated at 37° C. for3.5 hours. The final assay mixture (100 μl) contained 25 μMtri[1-¹⁴C]oleoylglycerol (250 000 DPM), 0.05% Triton X-100, and 50 mMimidazole-HCl (pH 7.0). The reaction was stopped and the lipid phaseextracted as in the steroid sulphatase assay described above forExample 1. The lower chloroform layer was dried and resuspended into 20μl chloroform:methanol (2:1) and loaded onto a preparative silica TLCplate. The lipids were developed with petroleum ether:diethylether:acetic acid (90:10:1) to the end of the plate (17 cm). The TLCplate was removed from the developing chamber and dried. The plate wasexposed to photographic film in a cassette for 3 days. The film wasremoved from the cassette and the image developed.

The results showed that lipase activity was present in the hair fibreextracts, with several new reaction products observed. The reactionproducts were identified as dioleoylglycerol, monooleoylglycerol andfree oleic acid. Consequently the hair enzyme had the ability tohydrolyse the triacylglycerol into glycerol and free fatty acids. Thelipid profile in control samples containing pre-boiled extract wassimilar to that of control samples containing substrate only. Thereforethe possibility of non-enzymatic artefact in the assays could be ruledout.

Example 3

Determination of the Presence of Protease in Hair Fibres

Hair fibres taken from approx. 5 mm from the scalps of male and femaleindividuals, aged 25-35, were cleaned, dried and then ground in liquidN₂, and homogenised with an Ystral Homogeniser (high shear) using a 10mm probe in extraction buffer (50 mM Citrate-Phosphate pH6.0, 2M NaCl).

Triton X-100 (2% stock) was added to the homogenate to a finalconcentration of 0.1%, and intermittently mixed by gentle agitation andincubated on ice for 1 hour. Large hair debris was allowed toprecipitate and the solution was centrifuged at 13000 rpm for 5 minutesto remove insoluble protein. The resulting supernatant, containingsoluble protein was dialysed (MWCO ca 14000 kDa) in 50 mMcitrate-phosphate, pH 6.0. Glycerol was added to the dialysed extract to20% (v/v). The extracts were then aliquotted and stored at −20° C. untilrequired.

The protease catalysed hydrolysis of casein was determined using theEnzCheck Protease Kit (Molecular Probes) with BODIPY FL-casein assubstrate. In this reagent, the casein is heavily labelled with thefluorescent dye BODIPY-FL, causing autoquenching of the fluorescence.Upon hydrolysis into peptides, the quenching is eliminated, causing aconcomitant increase in fluorescence. Casein serves as a relatively nonspecific substrate to different classes of proteases and was used inorder to observe the overall protease activity in hair extracts.

In a typical assay, sample protein (approx. 10 g) was added to a finalreaction volume of 100 l, containing: 50 mM Tris-HCl pH 7.8 and 10-20 gBODIPY-casein. The reactions were initiated with the addition ofsubstrate and incubated at 30° C. After 30 minutes, the reactions wereterminated with the addition of 1 ml 7.5% acetic acid. The fluorescenceof the terminated reaction was measured using a Spex Fluoromaxfluorimeter set to 503 nm (excitation) and 510 nm (emission). Thefluorescence of each sample was measured at these wavelengths with 1second increments over a period of 10 seconds.

The results showed that caseinolytic activity was present in the hairfibre extracts.

The effect of pH on caseinolysis was examined using the followingbuffered assay systems: citrate-phosphate (pH4-7), tris-HCl (pH 7-9) andglycine-NaOH (pH 9-11). Two peaks of protease activity were observed atpH 5.5 and pH 8.0.

The pH profile allowed the identification of two main groups of proteaseactivity. The peaks of protease activity at pH 5.5 and 8.0 wasindicative of cysteine and serine proteases, respectively.

With the above information, it was decided to investigate the proteaseprofile of the hair extracts with the use of more specific peptidesubstrates (peptidyl-7-amido-4-methylcoumarin (peptidyl-AMC)substrates). Seven peptidyl-AMC substrates were used to study theprotease types within the hair extract. These were selected on the basisof their specificity for individual protease species.

Stock solutions of each peptidyl-AMC substrate (Bachem) were prepared at10 mM in DMSO and stored in aliquots at 4° C. Each aliquot was dilutedto 1 mM with MQ water, prior to use. All assays (100 μl final volume)were routinely conducted in triplicate. For the measurement of cysteineprotease activity, the standard assay contained 50 mM citrate-phosphatebuffer pH 6.0, 100 μM peptidyl-AMC, 5 mM cysteine and approximately 1 μgprotein extract. The assays were initiated by the addition of substrateand incubated at 30° C. for 2 hours. The reactions were terminated withthe addition of 1 ml ‘stop’ solution (100 mM sodium monochloroacetate,30 mM sodium acetate, 70 mM acetic acid).

The hydrolysed AMC was measured with the use of a fluorimeter set to 370nm (excitation) and 460 nm (emission). Protease activity was quantifiedusing standard concentrations of AMC. The fluorescence of each substrateand the hair extract were also measured and these values subtracted fromthe final fluorescence of each reaction, to give the value offluorescence increase due to AMC liberation.

The results showed that of the various specific peptide substratestested, the substrate for cathepsin L, namely z-Phe-Arg-AMC, was foundto be the most hydrolysed by the hair extracts. Further tests showedthat the activity was stimulated by cysteine, potently inhibited byE-6464 (L-trans-epoxysuccinyl-leucylamido(4-guanido)butane), and mostactive under acidic conditions at 37° C., thereby confirming theactivity to be that of a cathepsin-L type protease. Cathepsin-L is alysosomal enzyme that is considered to have a broad specificity, and hasbeen shown to hydrolyse many cytosolic and structural proteins,including collagen and elastin. The apparent V_(max) of cathepsin L-typeactivity was calculated to be 1.66 nmoles AMC produced per minute per mghair extract (1 mg of extract is obtained from approx. 9 g hair).

No increase in fluorescence was observed in samples that had been boiledfor 20 minutes prior to assaying.

Example 4

Determination of the Presence of Transglutaminase (TGase) in Hair Fibres

Hair fibre clippings of 1-2 mm in length were obtained from within 5 mmof the scalp. Aliquots of 5 mg were placed into a conical, screw-cappedmicrofuge tube (Jencons) and 20 μl of 20 mM cystamine (a TGaseinhibitor) or 20 μl of water added. Samples were then made up to 50 μlwith water. To this 50 μl of a 2× solution of assay mixture containing200 mM Tris/HCl pH 8.5, 10 mM CaCl2, 20 mM DTT, 1% (w/v) TX-100 and 2μCi/ml [14C]-putrescine (118 Ci/mol stock) was added.

Samples were incubated at 37° C. for 1 hr and reactions stopped by theaddition of 10% (w/v) trichloroacetic acid (TCA). Fibres were recoveredby centrifugation at 13,000 rpm for 10 sec and washed three times with 1ml of TCA at 20 min intervals between incubations at 95° C. withshaking. The fibres were then solubilised by the addition of 20 μl of90% (w/v) toluene (Packard) and incubated at 95° C. with shaking untilcomplete dissolution had occurred (usually after about 10 min). Afteraddition of 1 ml of ReadySafe scintillation cocktail (Beckman) the tubeswere placed into small scintillation vials and the incorporation ofradioactive putrescine was measured in a Beckman LS 6000IC scintillationcounter.

Data were corrected by subtraction of non-specific backgroundrepresented by the samples containing cystamine. The results showed thatTGase activity was present in the hair fibre extracts.

Example 5

Determination of the Presence of Esterase in Hair Fibres

Unfixed cryosections of hair fibres were prepared and used to detectesterase activity. The assay mixture consisted of 50 mM Tris/HCl, pH7.5, 100 μM fluorescein diacetate (FDA). FDA is a recognised esterasesubstrate (Molecular Probes Handbook) and was made up as a 10 mM stockin dimethylsulphoxide and diluted into the assay mixture.

100 μl of assay mixture or 100 μl of 50 mM Tris/HCl, pH7.5 was placedonto the slides and overlaid with a glass coverslip. The slides wereincubated at room temperature for 30 min before detection of activity byfluorescence microscopy. Photographs were taken with a digital camerawith the gain set to give no signal in the sections treated with Trisbuffer alone (i.e. the negative control). The results showed thatesterase activity was present in the hair fibre extracts.

What is claimed is:
 1. A process for treating hair comprising applyingto said hair cholesterol-3-sulphate in an amount effective to activatehair fibre steroid sulphatase.